EP0728198B1 - Nukleinsäure-isolierung - Google Patents

Nukleinsäure-isolierung Download PDF

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Publication number
EP0728198B1
EP0728198B1 EP95900235A EP95900235A EP0728198B1 EP 0728198 B1 EP0728198 B1 EP 0728198B1 EP 95900235 A EP95900235 A EP 95900235A EP 95900235 A EP95900235 A EP 95900235A EP 0728198 B1 EP0728198 B1 EP 0728198B1
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EP
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Prior art keywords
sample
nucleic acid
boiling
support
solid support
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EP95900235A
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English (en)
French (fr)
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EP0728198A1 (de
Inventor
Jarle Breivik
Gustav Gaudernack
Anne Spurkland
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Medinnova SF
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Qiagen AS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation

Definitions

  • the present invention relates to the isolation of nucleic acid, especially DNA, and in particular to a method for preparing nucleic acid samples for subsequent use in amplification procedures.
  • DNA-based diagnostic techniques relying on DNA amplification, e.g. by the polymerise chain reaction (PCR), to detect the presence of microbial genes have proved useful in detecting bacterial and viral infectious agents and pathogenes and are rapidly acquiring importance.
  • PCR polymerise chain reaction
  • the techniques are not suitable for all diagnostic uses.
  • One major problem is that amplification techniques such as PCR cannot be used directly on clinical samples, notably faeces or blood, which contain substances which inhibit the amplification enzymes, e.g. polymerases.
  • the presence of red blood cells or haemoglobin presents a particular problem, and these generally need to be removed.
  • a similar problem applies in the case of detecting microbial contamination in food samples, which also often contain inhibitory substances.
  • the sample either has to be diluted very many times or the DMA has to be isolated and purified from the sample.
  • dilution to a degree sufficient to permit amplification frequently entails an unacceptable loss of sensitivity.
  • nucleic acid purification techniques involving for example extraction with phenols, chloroform and alcohols are often tedious, complicated and time consuming and may lead to loss of sample DNA, which can be a problem if the sample is small.
  • PCR has also proved difficult to apply to samples which are old, which have been chemically treated (for example by fixing or embedding) or which are otherwise distressed. This significantly impairs the utility of the technique in for example the analysis of fixed archival material or of blood and tissue samples which are not fresh or which have not been stored under refrigeration.
  • Fixation techniques now generally used do not permit ready release of DNA suitable for the subsequent amplification using conventional techniques, and whilst improved, less damaging, fixation techniques are being developed, the situation is not entirely satisfactory.
  • Complicated treatment procedures e.g. deparaffinization of paraffin-embedded material, proteinase digestion etc are-generally required and in many cases amplification cannot be achieved at all. This applies also in the case of aged or non-refrigerated samples.
  • EP 0393744 describes a method of extracting, amplifying and detecting a nucleic acid from whole blood or a peripheral blood mononuclear cell (PBMC) fraction thereof. Extraction from the PBMC fraction is accomplished by heating the fraction at or near the boiling point of water for a few minutes and then recovering the extracted nucleic acid. Whole blood can likewise be heated after it is mixed with a salt solution containing a polysaccharide, such as dextran. The extracted nucleic acid is then recovered from the heated mixture.
  • EP 0389063 describes a process for isolating nucleic acid from a nucleic acid-containing starting material such as whole blood, blood serum, urine, faeces, and cell cultures.
  • This method is characterized by mixing the starting material with a chaotropic substance and a nucleic acid binding solid phase.
  • the solid phase with the nucleic acid bound is then separated from the liquid.
  • the solid phase-nucleic acid complexes obtained are washed and if required the nucleic acid can be eluted from said complexes.
  • nucleic acid may be isolated from a sample in a form directly suitable for amplification by a simple and easy to perform procedure which involves boiling or heating the sample to a high temperature and allowing it to cool, and depositing the nucleic acid onto a solid support.
  • This procedure avoids many of the , complicated and time-consuming treatment steps of the prior art and, more importantly, can successfully be directly applied to clinical and other blood-containing samples and to samples which are aged, non-refrigerated or fixed, where previous techniques have proved unsuccessful.
  • the invention is based on the surprising discovery that when a sample is treated in this manner, nucleic acid released is able to condense around the support, thereby allowing it to be separated from the sample, whilst at the same time, retaining the ability to act as a template in a subsequent amplification reaction.
  • the present invention thus provides a method for isolating nucleic acid from a fixed or aged sample, said method comprising boiling said sample or heating said sample to a high temperature so as to release nucleic acid, and allowing it to cool, and binding the nucleic acid onto a high-surface area solid support.
  • a further aspect of the present invention provides a method for isolating nucleic acid from a sample, said method comprising boiling said sample or heating said sample to a high temperature so as to release nucleic acid and allowing it to cool, and binding the nucleic acid onto a high-surface area solid support comprising magnetic particles.
  • this method has particular utility in the preparation of nucleic acid samples for amplification procedures since the resulting condensed nucleic acid samples can be used directly as the template for amplification without requiring prior removal from the support.
  • the nucleic acid may be DNA, RNA or any modification thereof.
  • the nucleic acid will be DNA, which may be genomic or cDNA, and single or double stranded.
  • the method is used to prepare nucleic acid for amplification, it will preferably be double-stranded genomic DNA.
  • the sample may be any sample containing nucleic acid, but preferably will be a clinical sample such as a blood, blood-derived, faeces or tissue sample or a sample which is aged or treated.
  • Treated samples include those which have been fixed, for example in formalin, acetone, alcohols, or any known or proprietary fixative e.g. Omnifix, or embedded, for example in paraffin wax or artificial or natural resins.
  • Aged samples includes any sample which has not been freshly taken, or immediately refrigerated. Such aged or treated samples thus include any source of nucleic acid, plant or animal.
  • archival material which may be very many years old e.g.
  • samples therefore include samples of any biological tissue or fluid (e.g. blood, plasma, serum, organ or other tissue biopsies) which have not immediately been refrigerated or processed, e.g. clinical samples taken in remote areas which need to be transported and/or stored sometimes over a period of days, weeks or months, before they can be processed or analyzed.
  • biological tissue or fluid e.g. blood, plasma, serum, organ or other tissue biopsies
  • the present invention may be used when medical studies are conducted in remote regions of the world, where specialised equipment and personnel are lacking, and where samples require fixation and/or storage for extended periods of time before analysis.
  • the need for refrigeration may be avoided which is of significant benefit where samples need to be transported, e.g. by post.
  • the duration and temperature used in the boiling/heating step is to some extent dependent on the nature and state of the nucleic acid containing sample. Chemically treated, e.g. formalin treated, or aged samples generally require and can withstand more aggresive treatment than fresh cell suspensions. In general, the boiling/heating stage will conveniently be effected for 10 seconds to 1 hour, or more if convenient, for example 30 seconds to 30 minutes, or more particularly 3 to 15 minutes. Thus, for example, for most samples treatment at 94°C for 10 minutes will normally allow sufficient nucleic acid isolation to permit effective amplification.
  • the support may be present with the sample during the heat treatment or it may be introduced subsequently, even days or hours after the sample has been allowed to cool.
  • the support may be present with the sample during the heat treatment or it may be introduced subsequently, even days or hours after the sample has been allowed to cool.
  • it will generally be convenient to introduce the support before, during or shortly after the boiling/heating stage; however for samples where quantities of detritus separate out as a result of the boiling/heating stage it will generally be preferred to introduce the support after such detritus has been removed.
  • samples such as paraffin-embedded tissue
  • boiling/heating produces an inhomogeneous mixture containing tissue and wax fragments. It is preferred to separate off the nucleic acid containing liquid phase from these mixtures, e.g. by decanting or pipetting, after boiling/heating and optionally after cooling and then to introduce that liquid phase to the support to allow the nucleic acid condensation to occur.
  • the nucleic acid condensation (and preferably also the boiling/heating and cooling steps) in the method of the invention is preferably carried out in a high-salt aqueous solution (e.g. having an osmolality equivalent to that of at least 1 M NaCl aqueous solution).
  • a high-salt aqueous solution e.g. having an osmolality equivalent to that of at least 1 M NaCl aqueous solution.
  • condensation is effected by contacting the support with an already boiled/heated and cooled nucleic acid containing aqueous sample
  • contact is preferably made for a period of some minutes, e.g. 1 to 60, especially 10 to 20 minutes to allow the condensation, i.e. the binding of the nucleic acid to the support, to occur to an adequate extent.
  • the high-surface area support may be any known or conventional solid support presenting a high surface area for condensation of the nucleic acid.
  • Such supports will generally have an irregular surface, and may for example be porous or particulate eg. particles, fibres, webs, sinters, or sieves. Condensation of the nucleic acid may take place on or around the support, or in it, if it has a porous structure for example.
  • suitable solid supports include microtitre wells, capillaries, fibres, filters and dipsticks, although particular supports are generally preferred, especially beads, as they, with their captured, "condensed" nucleic acid, may readily be used directly in a subsequent amplification step without any need to detach the condensed nucleic acid from the support.
  • a particulate solid support used according to the invention will comprise spherical beads.
  • the size of the beads is not critical, but they may for example be of the order of diameter of at least 1 and preferably at least 2 ⁇ m, and have a maximum diameter of preferably not more than 10 and more preferably not more than 6 ⁇ m. Beads of diameter 2.8 ⁇ m and 4.8 ⁇ m have been shown to work well.
  • Monodisperse particles that is those which are substantially uniform in size (e.g. size having a diameter standard deviation of less than 5% have the advantage that they provide very uniform reproducibility of reaction.
  • Monodisperse polymer particles produced by the technique described in US-A-4336173 are especially suitable.
  • Non-magnetic polymer beads suitable for use in the method of the invention are available from Dyno Particles AS (Lillestrenn, Norway) as well as from Qiagen, Pharmacia and Serotec.
  • magnetic particles are particularly preferred as a solid support according to the invention, as they lend a number of advantages, Most notably, magnetic aggregation provides a quick, simple and efficient way of separating the particles following the condensation step, and is a far less rigorous method than traditional techniques such as centrifugation which generate shear forces which degrade nucleic acids.
  • Magnetic particles suitable for use as supports in the method of the invention are available from Dynal, Advanced Magnetics Inc., Biotechnologies Ltd., Amersham, Promega, Scigen, Advanced Genetic Technologies and Seradyn.
  • superparamagnetic particles for example those described by Sintef in WO-A-83/03920, as magnetic aggregation and clumping of the particles during reaction can be avoided, thus ensuring uniform and nucleic acid abstraction.
  • oligonucleotide probes specific for particular genes or nucleotide sequences may additionally be used, optionally together with stringent washing.
  • a specific probe may be introduced into the sample, binding to a specific target sequence.
  • the solid support may be provided with means for capture of the probe. Generally, this will be accomplished by providing each of the probe and the support with one of a pair of corresponding affinity binding partners, such that the probe and the support may be bound together selectively, and if desired, reversibly.
  • affinity binding partner will comprise biotin and avidin/streptavidin, the biotin being bound to the probe and the avidin/streptavidin to the support.
  • biotin-labelled oligonucleotide probes and streptavidin-coated magnetic particles are commercially available (Dynal AS).
  • binding partner systems may however be used, for example DNA-binding proteins binding to specific regions of the oligonucleotide probe e.g. the lac repressor protein Lac I binding to a lac operator ( Lac OP) site which may be provided on the probe.
  • the oligonucleotide probe may be labelled with a hapten such as digoxigenin, and the support may be provided with an anti-hapten antibody.
  • hapten such as digoxigenin
  • Techniques for labelling oligonucleotides with haptens such as digoxigenin are well known in the art as are methods for attachment of antibodies to solid supports.
  • Such a system is especially suited to the preparation of samples containing specific target nucleic acid sequences of interest, for amplification reactions.
  • prior art methods based upon isolation of target DMA for PCR by the use of specific biotin-labelled probes bound to streptavidin-coated magnetic beads have failed, e.g. in the case of blood containing samples, or aged or fixed samples, the method of the present invention has worked successfully.
  • the present invention thus provides a method of preparing a nucleic acid sample for in vitro amplification, said method comprising, sequentially or simultaneously, contacting said sample with an oligonucleotide probe specific for a target nucleotide sequence within said sample, and isolating nucleic acid from said sample by a method described above, wherein each of the oligonucleotide probe and solid support is provided with one of a pair of affinity binding partners, whereby the probe, and hence the target nucleotide sequence is bound to the support.
  • this aspect of the invention provides a method of amplification of nucleic acid within a sample, said method comprising the steps of (a) sequentially or simultaneously, contacting said sample with an oligonucleotide probe specific for a target nucleotide sequence within said sample, and isolating nucleic acid from said sample by a method described above, wherein each of the oligonucleotide probe and solid support is provided with one of a pair of affinity binding partners, whereby the probe, and hence the target nucleotide sequence is bound to the support, and (b) subjecting nucleic acid isolated by step (a) to an in vitro amplification reaction.
  • the sample may be allowed to cool in the presence of a solid support, whereby the nucleic acid condenses on or in the support.
  • the affinity binding partner pair will comprise biotin/streptavidin.
  • This binding partner system is commonly used in molecular biology applications and many methods of incorporating or attaching biotin and streptavidin to the probe/support respectively, are known.
  • biotin may be incorporated by and streptavidin may be coated onto the support as discussed by Dynal AS in their "Technical handbook, molecular biology”.
  • the length of the oligonucleotide probe is not critical but may conveniently lie in the range of 10-200 nucleotides, more preferably 15-50 nucleotides.
  • the functionalisation of the particles and subsequent attachment of probes is conveniently such that each particle carries 10 3 -10 6 probes or probe binding sites.
  • two primers are required and these may either be specific to a target DNA sequence of interest, or one or two standard PCR primers. This may necessitate introducing a hybridisation site for a standard PCR primer according to techniques well known in the art e.g. by restriction and ligation.
  • Nested PCR involves the use of two further so-called “inner” primers, which hybridise or “nest” between the first "outer” primer pair in a second series of amplification cycles.
  • the use of four separate priming events results in increased specificity of the amplification reaction.
  • the nested primer technique has further been modified in the DIANA (Detection of Immobilised Amplified Nucleic Acids) system (see Wahlberg et al ., Mol. Cell Probes 4 : 285(1990)), in which the inner, second pair of primers carry, respectively, means for immobilisation to permit capture of amplified DNA, and a label or means for attachment of a label to permit recognition.
  • DIANA Detection of Immobilised Amplified Nucleic Acids
  • the inner, second pair of primers carry, respectively, means for immobilisation to permit capture of amplified DNA, and a label or means for attachment of a label to permit recognition.
  • SSR Self-sustained Sequence Replication
  • LAR Ligase Amplification Reaction
  • primers are used which carry polymerase binding sites permitting the action of reverse transcriptase to amplify target RNA or ssDNA.
  • an immobilized probe captures one strand of target DNA and is then caused to hybridise with an RNA probe which carries as a template region a tertiary structure known as MDV-1 for an RNA-directed RNA polymerase, normally Q-beta replicase.
  • LAR hybridises two oligonucleotide probes to adjacent positions on the target nucleic acid so that ligation, e.g. using T4 ligase, produces a longer sequence, which after strand separation, can function as a template for further hybridisations and ligations.
  • the order in which the various steps are performed is not critical and variations and modification are possible.
  • the solid support may be added to the sample prior to boiling/heating, or after the boiling/heating step.
  • the primers/probes required for amplification may likewise be added prior to the boiling step, immediately after boiling/heating, or after the cooling condensation step.
  • the use of magnetic particles as solid supports particularly facilitates the washing and separation steps and has a significant advantage in that all the reactions, including amplification, may be performed in one reaction vessel, thereby considerably simplifying the reaction process and avoiding loss of nucleic acid fragments adhering to the vessel walls.
  • Boiling/heating of the sample may take place in any known or conventional medium known in the art for manipulation of nucleic acids.
  • many typical buffers e.g. washing buffers, are known and can be used.
  • high salt e.g. 1-4M
  • Exemplary buffers include Tris-buffered saline solutions, and Dynal AS's Binding and Washing buffer (10 mM Tris-HCl, 1 mM EDTA, 2.0M NaCl pH 7.5) is particularly suitable.
  • Similar buffers may be used for any intermediate washing steps which may be required. It has been found favourable to include salt, e.g. 1 to 4 M NaCl, preferably 2M NaCl, in the boiling medium.
  • Incubation times for boiling/heating may vary between 10 seconds and several hours, more conveniently between 1 and 15 minutes.
  • favourable results have been achieved by heating samples to a temperature of 80 to 100°C for a period of 10 minutes, or in some cases, 3 to 5 minutes.
  • Cooling may take place simply by allowing the sample to stand at ambient temperature, for example for a period of 3 to 20 minutes.
  • sample volumes of 0.1 ⁇ L to 100 mL.
  • sample volumes of 1 to 100 ⁇ L, e.g. 10 to 20 ⁇ L are preferred.
  • amounts of 1 to 500 ⁇ g preferably 20 to 200 ⁇ g may conveniently be used.
  • the ability of the method of the invention to be performed with such small sample quantities is of particular benefit as the quantity of sample available is often very limited, e.g. with needle biopsy and forensic samples or where it is important to keep the sample for further study.
  • oligonucleotide probe e.g. 1 to 5 pmol may be used.
  • kits form a further aspect of the invention.
  • a kit would generally comprise the reagents needed to identify particular gene segments from any given sample type, and particular examples of such kits would be for selective amplification of ras or HLA genes or their mutations, e.g. K-ras and its 12/13 codon point mutations.
  • kits will include magnetic beads for nucleic acid condensation; aqueous medium, e.g.
  • oligonucleotide probes conjugated or conjugable to the beads e.g. biotinylated probes which are conjugable to streptavidin coated beads
  • the method of the invention has a number of uses and applications. These include for example the detection of pathogens, diseases and allelic variations.
  • Clinical samples may be screened for epidemiological information. A major proposed use is in the analysis of clinical samples taken at remote locations which are transported to reference laboratories; blood samples may for example be directly posted without refrigeration once nucleic acid condensation and plasma and cell fragment purging has taken place. Thus the samples may be treated on site by the simple boiling step to condense the nucleic acid on to a solid support prior to flushing off sample other remnants and subsequent transport.
  • the technique also has utility in forensic medicine, and in all disciplines where sample or specimen storage is required e.g. in zoology, botany, conservation and evolutionary biology, the study of biological diversity or ecological processes.
  • Binding & Washing buffer (10 mM Tris-HCl, pH 7.5, 1mM EDTA, 2.0 M NaCl) containing 3 pmol of a biotinylated oligonucleotide (e.g. 5'-B-ACTGA ATATA AACTT GTGGT AGTTG GACCT-3') complimentary to the 5'-end of the K-ras gene segment.
  • a biotinylated oligonucleotide e.g. 5'-B-ACTGA ATATA AACTT GTGGT AGTTG GACCT-3'
  • the tubes are incubated at 94°C for 5 minutes and the contents mixed twice on a vortex-mixer for 20 seconds during this incubation.
  • the liquid phase is pipetted off and mixed with 20 ⁇ g of streptavidin coated paramagnetic beads (Dynabeads® M-280 streptavidin, Dynal, Norway).
  • the mixture is left at ambient temperature for 15 minutes.
  • the beads are isolated by magnetic separation (Dynal MPC®-E, Dynal, Norway), and added as template to a K-ras specific PCR reaction-mix (e.g. using the 5' and 3' end primers 5'-ACTGA ATATA AACTT GTGGT AGTTG GACCT-3' and 5'-TCAAA GAATG GTCCT GGAAC-3', and Taq DNA polymerase) 5 ⁇ L of the boiled liquid, collected before the beads are added, is used as template in a parallel control procedure.
  • a K-ras specific PCR reaction-mix e.g. using the 5' and 3' end primers 5'-ACTGA ATATA AACTT GTGGT AGTTG GACCT-3' and 5'-TCAAA GAATG GTCCT GGAAC-3', and Taq DNA polymerase
  • a detectable amplification product (lane 2) is obtained from the PCR where beads carrying DNA served as template, but not when the bead free boiled liquid is used (lane 3).
  • the FCR-product is identified as mutated by RFLP, and the nucleotide sequence was determined by solid-phase sequencing ( Figure 1b indicating the presence of a point mutation in codon 12.
  • Binding & Washing buffer 10 mM Tris-HCl, pH 7.5,m 1mM EDTA, 2.0 M NaCl
  • a parallel control sample was prepared without beads.
  • the tubes were then incubated at 94°C for 10 minutes and cooled to ambient temperature.
  • the beads were isolated by magnetic separation (Dynal MPC®-E, Dynal, Norway), and added to a HLA-DQB specific PCR reaction-mix (e.g. using the 5' and 3' end primers DQ-AMP A: 5'-GCATG TGCTA CTTCA CCAAC G-Biotin 3 1 and DQ-AMP B:5'-CAGGT AGTTG TGTCT GCACA C-3', and Taq DNA polymerase). 5 ⁇ l of the sample solution prepared without beads, was used as template in a parallel control procedure.
  • a detectable amplification product (column 2) was obtained from the PCR where beads carrying DNA served as template, but not when the bead free boiled liquid was used (column 3).
  • the nucleotide sequence was determined by solid-phase sequencing ( Figure 2b).
  • the method of the invention may thus conveniently be effected using the following steps:

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Claims (17)

  1. Verfahren zur Isolierung von Nucleinsäure aus einer fixierten oder gealterten Probe, wobei das Verfahren das Kochen der Probe oder das Erhitzen der Probe auf eine hohe Temperatur zwecks Freisetzung von Nucleinsäure sowie das Abkühlenlassen und das Binden der Nucleinsäure an einen festen Träger mit großem Oberflächenbereich umfasst.
  2. Verfahren zur Isolierung von Nucleinsäure aus einer Probe, wobei das Verfahren das Kochen der Probe oder das Erhitzen der Probe auf eine hohe Temperatur zwecks Freisetzung von Nucleinsäure sowie das Abkühlenlassen und das Binden der Nucleinsäure an einen magnetische Partikel enthaltenden, festen Träger mit großem Oberflächenbereich umfasst.
  3. Verfahren wie im Anspruch 1 oder 2 beansprucht, wobei die Probe gekocht wird.
  4. Verfahren wie in einem der Ansprüche 1 bis 3 beansprucht, wobei die Nucleinsäure DNA ist.
  5. Verfahren wie in einem der Ansprüche 1 bis 4 beansprucht, wobei die Probe eine klinische Probe ist.
  6. Verfahren wie in einem der Ansprüche 1 bis 5 beansprucht, wobei die Probe fixiert ist.
  7. Verfahren wie in einem der Ansprüche 1 bis 6 beansprucht, wobei die Probe gealtert ist.
  8. Verfahren wie in einem der Ansprüche 1 bis 7 beansprucht, wobei der Schritt des Kochens/Erhitzens durch Erhitzen der Probe auf mindestens 80°C bewirkt wird.
  9. Verfahren wie im Anspruch 8 beansprucht, wobei die Probe 10 Sekunden bis 1 Stunde lang erhitzt wird.
  10. Verfahren wie in einem der Ansprüche 1 bis 9 beansprucht, wobei der Träger vor oder während des Schritts des Kochens/Erhitzens zur Probe hinzugefügt wird.
  11. Verfahren wie in einem der Ansprüche 1 bis 9 beansprucht, wobei der Träger nach dem Schritt des Kochens/Erhitzens zur Probe hinzugefügt wird.
  12. Verfahren wie in einem der Ansprüche 1 bis 11 beansprucht, wobei die Schritte des Kochens/Erhitzens und/oder Abkühlens und/oder der Kondensation in einer hoch salzigen wässrigen Lösung ausgeführt werden.
  13. Verfahren wie in einem der Ansprüche 1 bis 12 beansprucht, wobei der feste Träger teilchenförmig ist.
  14. Verfahren wie in einem der Ansprüche 1 bis 13 beansprucht, wobei der feste Träger magnetische Partikel umfasst.
  15. Verfahren wie in einem der Ansprüche 1 bis 14 beansprucht, weiters umfassend das Hinzufügen einer für Zielnucleinsäure spezifischen Oligonucleotidsonde zur Probe und das Versehen des Trägers mit Mitteln zum Einfangen der Sonde.
  16. Verfahren zur Herstellung einer Nucleinsäureprobe für in vitro-Amplifikation, wobei dieses Verfahren der Reihe nach oder gleichzeitig das In-Kontakt-Bringen der Probe mit einer für eine Zielnucleotidsequenz in dieser Probe spezifischen Oligonucleotidsonde und das Isolieren von Nucleinsäure aus der Probe durch ein Verfahren gemäß einem der Ansprüche 1 bis 14 umfasst, wobei die Oligonucleotidsonde und der feste Träger jeweils mit einem von einem Paar Affinitätsbindungspartner versehen werden, wodurch die Sonde und daher die Zielnucleotidsequenz an den Träger gebunden wird.
  17. Verfahren zur Amplifikation von Nucleinsäure in einer Probe, wobei dieses Verfahren die Schritte des (a) der Reihe nach erfolgenden oder gleichzeitigen In-Kontakt-Bringens der Probe mit einer für eine Zielnucleotidsequenz in dieser Probe spezifischen Oligonucleotidsonde und des Isolierens von Nucleinsäure aus der Probe durch ein Verfahren gemäß einem der Ansprüche 1 bis 14 umfasst, wobei die Oligonucleotidsonde und der feste Träger jeweils mit einem von einem Paar Affinitätsbindungspartner versehen werden, wodurch die Sonde und daher die Zielnucleotidsequenz an den Träger gebunden wird, sowie (b) des Aussetzens der durch Schritt (a) isolierten Nucleinsäure einer in vitro-Amplifikationsreaktion.
EP95900235A 1993-11-11 1994-11-10 Nukleinsäure-isolierung Expired - Lifetime EP0728198B1 (de)

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GB9323305 1993-11-11
GB939323305A GB9323305D0 (en) 1993-11-11 1993-11-11 Isoaltion of nucleic acid
PCT/GB1994/002469 WO1995013368A1 (en) 1993-11-11 1994-11-10 Isolation of nucleic acid

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EP0728198A1 EP0728198A1 (de) 1996-08-28
EP0728198B1 true EP0728198B1 (de) 2003-09-10

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EP (1) EP0728198B1 (de)
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AU (1) AU682074B2 (de)
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AU8113194A (en) 1995-05-29
GB9323305D0 (en) 1994-01-05
DE69433136D1 (de) 2003-10-16
AU682074B2 (en) 1997-09-18
ATE249516T1 (de) 2003-09-15
EP0728198A1 (de) 1996-08-28
DK0728198T3 (da) 2003-10-13
DE69433136T2 (de) 2004-07-08
US6090935A (en) 2000-07-18
WO1995013368A1 (en) 1995-05-18
ES2206485T3 (es) 2004-05-16

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